Herpes simplex viruses (HSVs) cause cold sores, eye and genital infections, neonatal infections and encephalitis. The virus establishes lifelong latent infections in sensory ganglia. The envelope contains eleven virus-encoded glycoproteins of which four are essential for virus entry. These are gD, the receptor binding protein, gB and a complex of gH and gL. Several cell molecules serve as entry receptors and in each case the receptor binds gD. Most strains can use two of these, HveA (HVEM) and nectin- 1 (HveC). Whereas HveA (a TNF receptor) is found most abundantly on T lymphocytes, nectin- 1 (a cell adhesion molecule) is abundant on epithelial and neuronal cells. The specific aims of this grant are: (1) to characterize the interaction between gD and nectin- 1; (2) to determine the role HSV entry receptors in a mouse model of virus infection; and (3) to carry out structure-function studies of HSV gB. In Aim 1, our goal is to increase our understanding of the interaction between gD and nectin-1 in vitro, and on cells. When gD binds HveA, it undergoes two conformational changes as revealed by crystallographic data. We hypothesize that at least one of these also occurs when gD binds nectin- 1 and this change may play a role in later steps of entry. Solution of the structure of the gD/nectin-1 complex will test this hypothesis and is a major goal of this project. These studies combined with targeted mutagenesis will enhance our understanding of this protein-protein interaction. Nectin-1 can interact in trans with itself as well as with other nectins at cellular adherens junctions. We speculate that gD acts as a ligand for nectin-1 at these junctions so that receptor is available for virus spread to the next cell. Although nectin-1 appears to be more abundant than HveA on epithelial and neuronal cells, the role of these two receptors in HSV pathogenesis is not known. We developed a panel of gD mutants with altered ability to use one or the other receptor. In Aim 2, we will test the ability of viruses carrying these mutations to infect and cause zosteriform disease in mice. Although gB, gH and gL are required for fusion of the viral envelope with the plasma membrane of the host cell, their role in this process is not understood. In Aim 3, we will carry out structure function studies of gB in order to clarify its role in virus entry. We will follow up on the observation that gB partitions into lipid rafts on cells at the time of entry. Ultimately, we want to define the cellular targets of this interaction. Recently, we cloned, expressed and purified large quantities of the gB ectodomain. Crystals of this protein diffract and we therefore propose to solve the structure of gB by X-ray crystallography. The studies in this grant may lead to development of new approaches for antiviral therapeutics based on targets of the HSV entry process. [unreadable] [unreadable] [unreadable]